Genetically Altered Anti-body Producing Cell Lines With Improved Antibody Characteristics

ABSTRACT

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. Cells may be selected for expression of activation-induced cytidine deaminase (AID), stimulated to produce AID, or manipulated to express AID for further enhancement of hypermutability. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 10/933,034, filed Sep.2, 2004, which claims benefit of U.S. Provisional Application60/500,071, filed Sep. 3, 2003, both of which are incorporated byreference herein in their entireties.

TECHNICAL FIELD OF THE INVENTION

The invention is related to the area of antibody maturation and cellularproduction. In particular, it is related to the field of mutagenesis.

BACKGROUND OF THE INVENTION

The use of antibodies to block the activity of foreign and/or endogenouspolypeptides provides an effective and selective strategy for treatingthe underlying cause of disease. In particular is the use of monoclonalantibodies (MAb) as effective therapeutics such as the FDA approvedReoPro (Glaser, V. (1996) Can ReoPro repolish tarnished monoclonaltherapeutics? Nat. Biotechnol. 14:1216-1217), an anti-platelet MAb fromCentocor; Herceptin (Weiner, L. M. (1999) Monoclonal antibody therapy ofcancer. Semin. Oncol. 26:43-51), an anti-Her2/neu MAb from Genentech;and Synagis (Saez-Llorens, X. E., et al. (1998) Safety andpharmacokinetics of an intramuscular humanized monoclonal antibody torespiratory syncytial virus in premature infants and infants withbronchopulmonary dysplasia. Pediat. Infect. Dis. J. 17:787-791), ananti-respiratory syncytial virus MAb produced by Medimmune.

Standard methods for generating MAbs against candidate protein targetsare known by those skilled in the art. Briefly, rodents such as mice orrats are injected with a purified antigen in the presence of adjuvant togenerate an immune response (Shield, C. F., et al. (1996) Acost-effective analysis of OKT3 induction therapy in cadaveric kidneytransplantation. Am. J. Kidney Dis. 27:855-864). Rodents with positiveimmune sera are sacrificed and splenocytes are isolated. Isolatedsplenocytes are fused to melanomas to produce immortalized cell linesthat are then screened for antibody production. Positive lines areisolated and characterized for antibody production. The direct use ofrodent MAbs as human therapeutic agents were confounded by the fact thathuman anti-rodent antibody (HARA) responses occurred in a significantnumber of patients treated with the rodent-derived antibody (Khazaeli,M. B., et al., (1994) Human immune response to monoclonal antibodies. J.Immunother. 15:42-52). In order to circumvent the problem of HARA, thegrafting of the complementarity determining regions (CDRs), which arethe critical motifs found within the heavy and light chain variableregions of the immunoglobulin (Ig) subunits making up theantigen-binding domain, onto a human antibody backbone found thesechimeric molecules are able to retain their binding activity to antigenwhile lacking the HARA response (Emery, S. C., and Harris, W. J.“Strategies for humanizing antibodies” In: ANTIBODY ENGINEERING C. A. K.Borrebaeck (Ed.) Oxford University Press, N.Y. 1995. pp. 159-183. Acommon problem that exists during the “humanization” of rodent-derivedMAbs (referred to hereon as HAb) is the loss of binding affinity due toconformational changes in the three-dimensional structure of the CDRdomain upon grafting onto the human Ig backbone (U.S. Pat. No. 5,530,101to Queen et al.). To overcome this problem, additional HAb vectors areusually needed to be engineered by inserting or deleting additionalamino acid residues within the framework region and/or within the CDRcoding region itself in order to recreate high affinity HAbs (U.S. Pat.No. 5,530,101 to Queen et al.). This process is a very time-consumingprocedure that involves the use of expensive computer modeling programsto predict changes that may lead to a high affinity HAb. In someinstances the affinity of the HAb is never restored to that of the MAb,rendering them of little therapeutic use.

Another problem that exists in antibody engineering is the generation ofstable, high-yielding producer cell lines that is required formanufacturing of the molecule for clinical materials. Several strategieshave been adopted in standard practice by those skilled in the art tocircumvent this problem. One method is the use of Chinese Hamster Ovary(CHO) cells transfected with exogenous Ig fusion genes containing thegrafted human light and heavy chains to produce whole antibodies orsingle chain antibodies, which are a chimeric molecule containing bothlight and heavy chains that form an antigen-binding polypeptide (Reff,M. E. (1993) High-level production of recombinant immunoglobulins inmammalian cells. Curr. Opin. Biotechnol. 4:573-576). Another methodemploys the use of human lymphocytes derived from transgenic micecontaining a human grafted immune system or transgenic mice containing ahuman Ig gene repertoire. Yet another method employs the use of monkeysto produce primate MAbs, which have been reported to lack a humananti-monkey response (Neuberger, M., and Gruggermann, M. (1997)Monoclonal antibodies. Mice perform a human repertoire. Nature386:25-26). In all cases, the generation of a cell line that is capableof generating sufficient amounts of high affinity antibody poses a majorlimitation for producing sufficient materials for clinical studies.Because of these limitations, the utility of other recombinant systemssuch as plants are currently being explored as systems that will lead tothe stable, high-level production of humanized antibodies (Fiedler, U.,and Conrad, U. (1995) High-level production and long-term storage ofengineered antibodies in transgenic tobacco seeds. Bio/Technology13:1090-1093).

Other factors that naturally contribute to antibody diversity are thephenomena of class switch recombination (CSR) and somatic hypermutation.Class switch recombination is a region-specific recombination at the DNAlevel that results in the substitution of one immunoglobulin heavy chainregion for another. Somatic hypermutation is the name of the phenomenonin which fully assembled immunoglobulin genes nevertheless undergomutation in the variable regions only. Somatic hypermutation is thoughtto promote affinity maturation in antibodies.

An enzyme that has been found to play a critical role in both CSR andsomatic hypermutation is activation-induced cytodine deaminase (“AID” or“AICDA”). Muramatsu et al. cloned the murine AID (SEQ ID NOs:43 and 44)(Muramatsu et al. (1999) J. Biol. Chem. 274(26):18470-18476), while thehuman AID (SEQ ID NOs:39 and 40) was cloned by Muto et al. (2000)Genomics 68:85-88). The mouse and human AID share 92% identity at theamino acid level, both containing 198 amino acids with a conservedcytodine deaminase motif. It is believed that AID acts to induce lesionsin the DNA (i.e., deamination of deoxycytidines leading to dU/dG pairs)(Petersen-Mahrt et al. (2002) Nature 418:99-104). AID appears to beexpressed only in stimulated B cells in germinal centers (Okazaki et al.(2002) Nature 416:340-345), and appears to be responsible for both CSR(Petersen et al. (2001) Nature 414:660-665) and somatic hypermutation(Yoshikawa et al. (2002) Science 296:2033-2036).

Revy et al. showed that human patients with a defect in the AID gene(hyper IgM syndrome, or HIGM2) lacked both CSR and somatic hypermutationactivity (Revy et al. (2000) Cell 102:565-575). Similarly, spleen cellsfrom AID^(−/−) mice failed to undergo somatic hypermutation or CSR whenstimulated in vitro (Muramatsu et al. (2000) Cell 102:553-563).

A method for generating diverse antibody sequences within the variabledomain that results in HAbs and MAbs with high binding affinities toantigens would be useful for the creation of more potent therapeutic anddiagnostic reagents respectively. Moreover, the generation of randomlyaltered nucleotide and polypeptide residues throughout an entireantibody molecule will result in new reagents that are less antigenicand/or have beneficial pharmacokinetic properties.

SUMMARY OF THE INVENTION

The invention described herein is directed to the use of random geneticmutation throughout an antibody structure in vivo and in vitro byblocking the endogenous mismatch repair (MMR) activity of a host celland stimulating the activity of AID, producing immunoglobulins thatencode biochemically active antibodies. The invention also relates tomethods for repeated in vivo and in vitro genetic alterations andselection for antibodies with enhanced binding and pharmacokineticprofiles.

In addition, the ability to develop genetically altered host cells thatare capable of secreting increased amounts of antibody will also providea valuable method for creating cell hosts for product development. Theinvention described herein is directed to the creation of geneticallyaltered cell hosts with increased antibody production via the blockadeof MMR and stimulation of AID.

The invention facilitates the generation of high affinity antibodies andthe production of cell lines with elevated levels of antibodyproduction. Other advantages of the present invention are described inthe examples and figures described herein.

The invention provides methods for generating genetically alteredantibodies (including single chain molecules) and antibody-producingcell hosts in vitro and in vivo, whereby the antibody possesses adesired biochemical property(ies), such as, but not limited to,increased antigen binding, increased gene expression, and/or enhancedextracellular secretion by the cell host. One method for identifyingantibodies with increased binding activity or cells with increasedantibody production is through the screening of MMR-defectiveantibody-producing cell clones that produce molecules with enhancedbinding properties or clones that have been genetically altered toproduce enhanced amounts of antibody product.

The antibody-producing cells suitable for use in the invention include,but are not limited to, rodent, primate, or human hybridomas orlymphoblastoids; mammalian cells transfected and expressing exogenous Igsubunits or chimeric single chain molecules; plant cells, yeast, orbacteria transfected and expressing exogenous Ig subunits or chimericsingle chain molecules.

Thus, the invention provides methods for making hypermutableantibody-producing cells by introducing a polynucleotide comprising adominant negative allele of a mismatch repair gene into cells that arecapable of producing antibodies. The cells that are capable of producingantibodies include cells that naturally produce antibodies, and cellsthat are engineered to produce antibodies through the introduction ofimmunoglobulin encoding sequences. Conveniently, the introduction ofpolynucleotide sequences into cells is accomplished by transfection.

The invention also provides methods for producing hybridoma cellsproducing high-affinity antibodies from in vitro immunizedimmunoglobulin-producing cells comprising: (a) combining peripheralblood cells comprising immunoglobulin-producing cells with animmunogenic antigen in vitro; (b) fusing the immunoglobulin-producingcells with myeloma cells to form parental hybridoma cells, wherein thehybridoma cells express a dominant negative allele of a mismatch repairgene; (c) performing a screen for expression of activation-inducedcytidine deaminase; (d) incubating the hybridoma cells to allow formutagenesis, thereby forming hypermutated hybridoma cells. The cells maybe further screened for cells that produce antibody that specificallybinds the immunizing antigen. The selected cells may also be manipulatedto inactivate the dominant negative allele of the mismatch repair geneto restabilize the genome of the cell. The selected cells may also bemanipulated to inactivate the expression of AID.

In certain embodiments of the in vitro immunization method, theimmunoglobulin-producing cell and/or the myeloma cell is naturallydeficient in mismatch repair such that, upon fusion, the resultinghybridoma cell is naturally deficient in mismatch repair. In such acase, when restabilizing the genome, the cells must be manipulated togenetically complement the deficiency by any method known in the art.For example, but not by way of limitation, if the MMR deficiency is dueto loss of an essential gene for mismatch repair, the gene may bereintroduced into the cell operably linked to expression controlsequences such that the normal MMR gene is replaced and MMR activity isrestored. The expression of the MMR gene may be under the control of aconstitutive or an inducible promoter. In other cases in which the MMRdefect is the expression of a dominant negative allele of the MMR gene,the genome may be complemented by inactivation of the MMR gene. Forexample, but not by way of limitation, the defective MMR allele may beknocked out in whole or in part by any means known to the skilledartisan, such that the allele no longer asserts a dominant negativeeffect on mismatch repair.

In other embodiments of the in vitro immunization method, the hybridomacells are manipulated to be MMR deficient. In certain embodiments, adominant negative allele of a mismatch repair gene is introduced intothe antibody-producing cell. In other embodiments, the dominant negativeallele of a mismatch repair gene is introduced into the myeloma cell. Inother embodiments, the dominant negative allele of a mismatch repairgene is introduced into the hybridoma cell. The introduction of thedominant negative allele of a mismatch repair gene may be by any meansknown in the art such as, but not limited to, transfection.

The invention also provides methods for producing hybridoma cellsproducing high-affinity antibodies from in vitro immunizedimmunoglobulin-producing cells comprising: (a) combining peripheralblood cells comprising immunoglobulin-producing cells with animmunogenic antigen in vitro; (b) fusing the immunoglobulin-producingcells with myeloma cells to form parental hybridoma cells; (c)performing a screen for expression of activation-induced cytidinedeaminase; (d) incubating the hybridoma cells to allow for mutagenesis,thereby forming hypermutated hybridoma cells. The cells may be furtherscreened for cells that produce antibody that specifically binds theimmunizing antigen. The selected cells may also be manipulated toinactivate the expression of AID.

The invention also provides methods for producing hybridoma cellsproducing high affinity antibodies from in vitro immunizedimmunoglobulin-producing cells comprising: (a) combining peripheralblood cells comprising immunoglobulin-producing cells with animmunogenic antigen in vitro; (b) fusing the immunoglobulin-producingcells with myeloma cells to form parental hybridoma cells, wherein thehybridoma cells express a dominant negative allele of a mismatch repairgene; (c) inducing expression of activation-induced cytidine deaminase;(d) incubating the hybridoma cells to allow for mutagenesis, therebyforming hypermutated hybridoma cells. The cells may be further screenedfor cells that produce antibody that specifically binds the immunizingantigen. The selected cells may also be manipulated to inactivate thedominant negative allele of the mismatch repair gene to restabilize thegenome of the cell. The selected cells may also be manipulated toinactivate the expression of AID.

In some embodiments the AID gene is introduced into theantibody-producing cell, myeloma cell or hybridoma cell operably linkedto expression control sequences such that AID is expressed in the cells.In certain embodiments, AID is operably linked to an inducible promoter.In some embodiments, once cells are selected for the desired phenotype,AID expression is turned off, by any means known in the art such as byinactivation of the AID by partially or completely knocking out thegene, by withdrawing the inducer of the inducible promoter, and thelike. In some embodiments, the antibody-producing cells, myeloma cells,and/or hybridoma cells may be further manipulated to be defective inmismatch repair. In some embodiments, this is accomplished byintroducing into the cell a dominant negative allele of a mismatchrepair gene. In other embodiments, this is accomplished by incubatingthe cell in a chemical inhibitor of mismatch repair as described in WO02/054856 (Nicolaides et al, filed Jan. 15, 2001). To restabilize thegenome of the cell, the dominant negative allele may be inactivated, or,in the case of chemical inhibition of MMR, the chemical inhibitor may bewithdrawn or diluted out, for example.

The invention also provides methods of making hypermutableantibody-producing cells by introducing a dominant negative mismatchrepair (MMR) gene such as PMS2 (preferably human PMS2), MLH1, PMS1,MSH2, or MSH2 into cells that are capable of producing antibodies. Thedominant negative allele of a mismatch repair gene may be a truncationmutation of a mismatch repair gene (preferably a truncation mutation atcodon 134, or a thymidine at nucleotide 424 of wild-type PMS2). Theinvention also provides methods in which mismatch repair gene activityis suppressed. This may be accomplished, for example, using antisensemolecules directed against the mismatch repair gene or transcripts.

Other embodiments of the invention provide methods for making ahypermutable antibody-producing cell by introducing a polynucleotidecomprising a dominant negative allele of a mismatch repair gene into afertilized egg of an animal. These methods may also include subsequentlyimplanting the eggs into pseudo-pregnant females whereby the fertilizedeggs develop into a mature transgenic animal. The mismatch repair genesmay include, for example, PMS2 (preferably human PMS2), MLH1, PMS1,MSH2, or MSH2. The dominant negative allele of a mismatch repair genemay be a truncation mutation of a mismatch repair gene (preferably atruncation mutation at codon 134, or a thymidine at nucleotide 424 ofwild-type PMS2).

The invention further provides homogeneous compositions of cultured,hypermutable, mammalian cells that are capable of producing antibodiesand contain a dominant negative allele of a mismatch repair gene. Themismatch repair genes may include, for example, PMS2 (preferably humanPMS2), MLH1, PMS1, MSH2, or MSH2. The dominant negative allele of amismatch repair gene may be a truncation mutation of a mismatch repairgene (preferably a truncation mutation at codon 134, or a thymidine atnucleotide 424 of wild-type PMS2). The cells of the culture may containPMS2, (preferably human PMS2), MLH1, or PMS1; or express a human mutLhomolog, or the first 133 amino acids of hPMS2.

The invention further provides methods for generating a mutation in animmunoglobulin gene of interest by culturing an immunoglobulin-producingcell selected for an immunoglobulin of interest wherein the cellcontains a dominant negative allele of a mismatch repair gene. Theproperties of the immunoglobulin produced from the cells can be assayedto ascertain whether the immunoglobulin gene harbors a mutation. Theassay may be directed to analyzing a polynucleotide encoding theimmunoglobulin, or may be directed to the immunoglobulin polypeptideitself.

The invention also provides methods for generating a mutation in a geneaffecting antibody production in an antibody-producing cell by culturingthe cell expressing a dominant negative allele of a mismatch repairgene, and testing the cell to determine whether the cell harborsmutations within the gene of interest, such that a new biochemicalfeature (e.g., over-expression and/or secretion of immunoglobulinproducts) is generated. The testing may include analysis of the steadystate expression of the immunoglobulin gene of interest, and/or analysisof the amount of secreted protein encoded by the immunoglobulin gene ofinterest. The invention also embraces prokaryotic and eukaryotictransgenic cells made by this process, including cells from rodents,non-human primates, and humans.

Other aspects of the invention encompass methods of reversibly alteringthe hypermutability of an antibody-producing cell, in which an induciblevector containing a dominant negative allele of a mismatch repair geneoperably linked to an inducible promoter is introduced into anantibody-producing cell. The cell is treated with an inducing agent toexpress the dominant negative mismatch repair gene (which can be PMS2(preferably human PMS2), MLH1, or PMS1). Alternatively, the cell may beinduced to express a human mutL homolog or the first 133 amino acids ofhPMS2. In another embodiment, the cells may be rendered capable ofproducing antibodies by co-transfecting a preselected immunoglobulingene of interest. The immunoglobulin genes of the hypermutable cells, orthe proteins produced by these methods may be analyzed for desiredproperties, and induction may be stopped such that the genetic stabilityof the host cell is restored.

The invention also embraces methods of producing genetically alteredantibodies by transfecting a polynucleotide encoding an immunoglobulinprotein into a cell containing a dominant negative mismatch repair gene(either naturally or in which the dominant negative mismatch repair genewas introduced into the cell), culturing the cell to allow theimmunoglobulin gene to become mutated and produce a mutantimmunoglobulin, screening for a desirable property of the mutantimmunoglobulin protein, isolating the polynucleotide molecule encodingthe selected mutant immunoglobulin possessing the desired property, andtransfecting said mutant polynucleotide into a genetically stable cell,such that the mutant antibody is consistently produced without furthergenetic alteration. The dominant negative mismatch repair gene may bePMS2 (preferably human PMS2), MLH1, or PMS1. Alternatively, the cell mayexpress a human mutL homolog or the first 133 amino acids of hPMS2.

The invention further provides methods for generating geneticallyaltered cell lines that express enhanced amounts of an antigen-bindingpolypeptide. These antigen-binding polyeptides may be, for example,immunoglobulins. The methods of the invention also include methods forgenerating genetically altered cell lines that secrete enhanced amountsof an antigen-binding polypeptide. The cell lines are renderedhypermutable by dominant negative mismatch repair genes that provide anenhanced rate of genetic hypermutation in a cell producingantigen-binding polypeptides such as antibodies. Such cells include, butare not limited to, hybridomas. Expression of enhanced amounts ofantigen-binding polypeptides may be through enhanced transcription ortranslation of the polynucleotides encoding the antigen-bindingpolypeptides, or through the enhanced secretion of the antigen-bindingpolypeptides, for example.

Methods are also provided for creating genetically altered antibodies invivo by blocking the MMR activity of the cell host, or by transfectinggenes encoding for immunoglobulin in a MMR-defective cell host.

Antibodies with increased binding properties to an antigen due togenetic changes within the variable domain are provided in methods ofthe invention that block endogenous MMR of the cell host. Antibodieswith increased binding properties to an antigen due to genetic changeswithin the CDR regions within the light and/or heavy chains are alsoprovided in methods of the invention that block endogenous MMR of thecell host.

The invention provides methods of creating genetically alteredantibodies in MMR defective Ab-producer cell lines with enhancedpharmacokinetic properties in host organisms including but not limitedto rodents, primates, and man.

These and other aspects of the invention are provided by one or more ofthe embodiments described below. In one embodiment of the invention, amethod for making an antibody-producing cell line hypermutable isprovided. A polynucleotide encoding a dominant negative allele of a MMRgene is introduced into an antibody-producing cell. The cell becomeshypermutable as a result of the introduction of the gene.

In another embodiment of the invention, a method is provided forintroducing a mutation into an endogenous gene encoding for animmunoglobulin polypeptide or a single chain antibody. A polynucleotideencoding a dominant negative allele of a MMR gene is introduced into acell. The cell becomes hypermutable as a result of the introduction andexpression of the MMR gene allele. The cell further comprises animmunoglobulin gene of interest. The cell is grown and tested todetermine whether the gene encoding for an immunoglobulin or a singlechain antibody of interest harbors a mutation. In another aspect of theinvention, the gene encoding the mutated immunoglobulin polypeptide orsingle chain antibody may be isolated and expressed in a geneticallystable cell. In a preferred embodiment, the mutated antibody is screenedfor at least one desirable property such as, but not limited to,enhanced binding characteristics.

In another embodiment of the invention, a gene or set of genes encodingfor Ig light and heavy chains or a combination thereof are introducedinto a mammalian cell host that is MMR-defective. The cell is grown, andclones are analyzed for antibodies with enhanced bindingcharacteristics.

In another embodiment of the invention, methods are provided forproducing new phenotypes of a cell. A polynucleotide encoding a dominantnegative allele of a MMR gene is introduced into a cell. The cellbecomes hypermutable as a result of the introduction of the gene. Thecell is grown and tested for the expression of new phenotypes, such asenhanced secretion of a polypeptide.

These and other embodiments of the invention provide the art withmethods that can generate enhanced mutability in cells and animals aswell as providing cells and animals harboring potentially usefulmutations for the large-scale production of high affinity antibodieswith beneficial pharmacokinetic profiles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates hybridoma cells stably expressing PMS2 and PMS134 MMRgenes. Shown is steady state mRNA expression of MMR genes transfectedinto a murine hybridoma cell line. Stable expression was found after 3months of continuous growth. The (−) lanes represent negative controlswhere no reverse transcriptase was added, and the (+) lanes representsamples reverse-transcribed and PCR-amplified for the MMR genes and aninternal housekeeping gene as a control.

FIG. 2 shows the creation of genetically hypermutable hybridoma cells.Dominant negative MMR gene alleles were expressed in cells expressing aMMR-sensitive reporter gene. Dominant negative alleles such as PMS134and the expression of MMR genes from other species results inantibody-producer cells with a hypermutable phenotype that can be usedto produce genetically altered immunoglobulin genes with enhancedbiochemical features as well as lines with increased Ig expressionand/or secretion. Values shown represent the amount of converted CPRGsubstrate which is reflective of the amount of function β-galactosidasecontained within the cell from genetic alterations within the pCAR-OFreporter gene. Higher amounts of β-galactosidase activity reflect ahigher mutation rate due to defective MMR.

FIG. 3 illustrates a screening method for identifying antibody-producingcells containing antibodies with increased binding activity and/orincreased expression/secretion

FIG. 4 illustrates the generation of a genetically altered antibody withan increased binding activity. Shown are ELISA values from 96-wellplates screened for antibodies specific to hIgE. Two clones with a highbinding value were found in HB134 cultures.

FIG. 5A illustrates sequence alteration within variable chain of anantibody (a mutation within the light chain variable region inMMR-defective HB134 antibody-producer cells). Arrows indicate thenucleotide at which a mutation occurred in a subset of cells from aclone derived from HB134 cells. The HB134 sequence (SEQ ID NO:51) isshown as the top line and the parental H36 sequence (SEQ ID NO:52) isshown above and below the sequence tracing. The change results in a Thrto Ser change within the light chain variable region. The codingsequence is in the antisense direction.

FIG. 5B illustrates sequence alteration within variable chain of anantibody (a mutation within the light chain variable region inMMR-defective HB134 antibody-producer cells). The HB134 sequence (SEQ IDNO:53) is shown above and below the tracing for the HB134 sequence, andthe parental H36 sequence (SEQ ID NO:54) is shown above and below theH36 sequence tracing. A consensus sequence (SEQ ID NO:55) is shown atthe bottom of the figure. Arrows indicate the nucleotide at which amutation occurred in a subset of cells from a clone derived from HB 134cells. The change results in a Pro to Leu change within the light chainvariable region.

FIG. 6 illustrates the generation of MMR-defective clones with enhancedsteady state Ig protein levels. A Western blot of heavy chainimmunglobulins from HB134 clones with high levels of MAb (>500 ngs/ml)within the conditioned medium shows that a subset of clones expresshigher steady state levels of immunoglobulins (Ig). The H36 cell linewas used as a control to measure steady state levels in the parentalstrain. Lane 1: fibroblast cells (negative control); Lane 2: H36 cell;Lane 3: HB134 clone with elevated MAb levels; Lane 4: HB134 clone withelevated MAb levels; Lane 5: HB134 clone with elevated MAb levels.

FIG. 7 demonstrates the expression by selected clones ofactivation-induced cytidine deaminase. Lane 1, water control; Lane 2,5-8 RT+ (SUPERSCRIPT); Lane 3, 5-8 RT− (SUPERSCRIPT); Lane 4, 7-6(EXPRESSDIRECT); Lane 5, 8-2 (EXPRESSDIRECT); Lane 6, 3-32(EXPRESSDIRECT).

DETAILED DESCRIPTION OF THE INVENTION

The reference works, patents, patent applications, and scientificliterature, including accession numbers to GenBank database sequencesthat are referred to herein establish the knowledge of those with skillin the art and are hereby incorporated by reference in their entirety tothe same extent as if each was specifically and individually indicatedto be incorporated by reference. Any conflict between any referencecited herein and the specific teachings of this specification shall beresolved in favor of the latter. Likewise, any conflict between anart-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,New York (1998); Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL,2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989);Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND C ELLULAR METHODS INBIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).

As used herein “activating cytokine” means a soluble molecule thatstimulates cells to express a new protein, differentiate or proliferate.

As used herein, the term “epitope” refers to the portion of an antigento which a monoclonal antibody specifically binds.

As used herein, the term “conformational epitope” refers to adiscontinuous epitope formed by a spatial relationship between aminoacids of an antigen other than an unbroken series of amino acids.

As used herein, the term “isoform” refers to a specific form of a givenpolypeptide.

As used herein, the term “immunobased” refers to protein-based therapiesto generate immunological responses that can specifically orpreferentially kill target bearing cells.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism.

The term “therapeutic effect” refers to the inhibition of an abnormalcondition. A therapeutic effect relieves to some extent one or more ofthe symptoms of the abnormal condition. In reference to the treatment ofabnormal conditions, a therapeutic effect can refer to one or more ofthe following: (a) an increase or decrease in the proliferation, growth,and/or differentiation of cells; (b) inhibition (i.e., slowing orstopping) of growth of tumor cells in vivo (c) promotion of cell death;(d) inhibition of degeneration; (e) relieving to some extent one or moreof the symptoms associated with the abnormal condition; and (f)enhancing the function of a population of cells. The monoclonalantibodies and derivatives thereof described herein effectuate thetherapeutic effect alone or in combination with conjugates or additionalcomponents of the compositions of the invention.

As used herein, the term “about” refers to an approximation of a statedvalue within an acceptable range. Preferably the range is +/−5% of thestated value.

As used herein “dominant negative effect” refers to the ability of anallele of a mismatch repair gene to inhibit normal mismatch repair incells, which may be assessed by the cells exhibiting microsatelliteinstability.

Stimulation of expression includes any means of increasing theexpression of a nucleic acid sequence or a peptide and includes but isnot limited to stimulation of endogenous expression; inducibleexpression; inserting a constitutively active promoter, etc.

As used herein, “mitogenic polypeptide” refers to a polypeptide that maybe conjugated to an immunogen to enhance stimulation of the immunesystem to the antigen.

As used herein “cells capable of producing antibodies” refers to cellsthat are naturally capable of producing immunoglobulins. Sources forsuch cells are, for example, lymph node cells, spleen cells, peripheralblood cells, and antibody-producing cell lines.

Methods have been discovered for developing hypermutableantibody-producing cells by taking advantage of the conserved mismatchrepair (MMR) process of host cells. Dominant negative alleles of suchgenes, when introduced into cells or transgenic animals, increase therate of spontaneous mutations by reducing the effectiveness of DNArepair and thereby render the cells or animals hypermutable.Hypermutable cells or animals can then be utilized to develop newmutations in a gene of interest. Blocking MMR in antibody-producingcells (such as but not limited to: hybridomas; mammalian cellstransfected with genes encoding for Ig light and heavy chains; mammaliancells transfected with genes encoding for single chain antibodies;eukaryotic cells transfected with Ig genes) can enhance the rate ofmutation within these cells leading to clones that have enhancedantibody production and/or cells containing genetically alteredantibodies with enhanced biochemical properties such as increasedantigen binding. The process of MMR, also called mismatch proofreading,is carried out by protein complexes in cells ranging from bacteria tomammalian cells. A MMR gene is a gene that encodes for one of theproteins of such a mismatch repair complex. Although not wanting to bebound by any particular theory of mechanism of action, a MMR complex isbelieved to detect distortions of the DNA helix resulting fromnon-complementary pairing of nucleotide bases. The non-complementarybase on the newer DNA strand is excised, and the excised base isreplaced with the appropriate base, which is complementary to the olderDNA strand. In this way, cells eliminate many mutations that occur as aresult of mistakes in DNA replication.

Dominant negative alleles cause a MMR defective phenotype even in thepresence of a wild-type allele in the same cell. An example of adominant negative allele of a MMR gene is the human gene hPMS2-134,which carries a truncating mutation at codon 134 (SEQ ID NO:5). Themutation causes the product of this gene to abnormally terminate at theposition of the 134th amino acid, resulting in a shortened polypeptidecontaining the N-terminal 133 amino acids. Such a mutation causes anincrease in the rate of mutations, which accumulate in cells after DNAreplication. Expression of a dominant negative allele of a mismatchrepair gene results in impairment of mismatch repair activity, even inthe presence of the wild-type allele. Any allele which produces sucheffect can be used in this invention. Dominant negative alleles of a MMRgene can be obtained from the cells of humans, animals, yeast, bacteria,or other organisms. Such alleles can be identified by screening cellsfor defective MMR activity. Cells from animals or humans with cancer canbe screened for defective mismatch repair. Cells from colon cancerpatients may be particularly useful. Genomic DNA, cDNA, or mRNA from anycell encoding a MMR protein can be analyzed for variations from the wildtype sequence. Dominant negative alleles of a MMR gene can also becreated artificially, for example, by producing variants of thehPMS2-134 allele or other MMR genes. Various techniques of site-directedmutagenesis can be used. The suitability of such alleles, whethernatural or artificial, for use in generating hypermutable cells oranimals can be evaluated by testing the mismatch repair activity causedby the allele in the presence of one or more wild-type alleles, todetermine if it is a dominant negative allele.

A cell or an animal into which a dominant negative allele of a mismatchrepair gene has been introduced will become hypermutable. This meansthat the spontaneous mutation rate of such cells or animals is elevatedcompared to cells or animals without such alleles. The degree ofelevation of the spontaneous mutation rate can be at least 2-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or1000-fold that of the normal cell or animal. The use of chemicalmutagens such as but limited to methane sulfonate, dimethyl sulfonate,06-methyl benzadine, MNU, ENU, etc. can be used in MMR defective cellsto increase the rates an additional 10 to 100 fold that of the MMRdeficiency itself.

According to one aspect of the invention, a polynucleotide encoding fora dominant negative form of a MMR protein is introduced into a cell. Thegene can be any dominant negative allele encoding a protein, which ispart of a MMR complex, for example, PMS2, PMS1, MLH1, or MSH2. Thedominant negative allele can be naturally occurring or made in thelaboratory. The polynucleotide can be in the form of genomic DNA, cDNA,RNA, or a chemically synthesized polynucleotide.

The polynucleotide can be cloned into an expression vector containing aconstitutively active promoter segment (such as but not limited to CMV,SV40, Elongation Factor or LTR sequences) or to inducible promotersequences such as the steroid inducible pIND vector (Invitrogen), wherethe expression of the dominant negative MMR gene can be regulated. Thepolynucleotide can be introduced into the cell by transfection.

According to another aspect of the invention, an immunoglobulin (Ig)gene, a set of Ig genes or a chimeric gene containing whole or parts ofan Ig gene can be transfected into MMR deficient cell hosts, the cell isgrown and screened for clones containing genetically altered Ig geneswith new biochemical features. MMR defective cells may be of human,primates, mammals, rodent, plant, yeast or of the prokaryotic kingdom.The mutated gene encoding the Ig with new biochemical features may beisolated from the respective clones and introduced into geneticallystable cells (i.e., cells with normal MMR) to provide clones thatconsistently produce Ig with the new biochemical features. The method ofisolating the Ig gene encoding Ig with new biochemical features may beany method known in the art. Introduction of the isolated polynucleotideencoding the Ig with new biochemical features may also be performedusing any method known in the art, including, but not limited totransfection of an expression vector containing the polynucleotideencoding the Ig with new biochemical features. As an alternative totransfecting an Ig gene, a set of Ig genes or a chimeric gene containingwhole or parts of an Ig gene into an MMR-deficient host cell, such Iggenes may be transfected simultaneously with a gene encoding a dominantnegative mismatch repair gene into a genetically stable cell to renderthe cell hypermutable.

Transfection is any process whereby a polynucleotide is introduced intoa cell. The process of transfection can be carried out in a livinganimal, e.g., using a vector for gene therapy, or it can be carried outin vitro, e.g., using a suspension of one or more isolated cells inculture. The cell can be any type of eukaryotic cell, including, forexample, cells isolated from humans or other primates, mammals or othervertebrates, invertebrates, and single-celled organisms such asprotozoa, yeast, or bacteria.

In general, transfection will be carried out using a suspension ofcells, or a single cell, but other methods can also be applied as longas a sufficient fraction of the treated cells or tissue incorporates thepolynucleotide so as to allow transfected cells to be grown andutilized. The protein product of the polynucleotide may be transientlyor stably expressed in the cell. Techniques for transfection are wellknown. Available techniques for introducing polynucleotides include butare not limited to electroporation, transduction, cell fusion, the useof calcium chloride, and packaging of the polynucleotide together withlipid for fusion with the cells of interest. Once a cell has beentransfected with the MMR gene, the cell can be grown and reproduced inculture. If the transfection is stable, such that the gene is expressedat a consistent level for many cell generations, then a cell lineresults.

An isolated cell is a cell obtained from a tissue of humans or animalsby mechanically separating out individual cells and transferring them toa suitable cell culture medium, either with or without pretreatment ofthe tissue with enzymes, e.g., collagenase or trypsin. Such isolatedcells are typically cultured in the absence of other types of cells.Cells selected for the introduction of a dominant negative allele of amismatch repair gene may be derived from a eukaryotic organism in theform of a primary cell culture or an immortalized cell line, or may bederived from suspensions of single-celled organisms.

A polynucleotide encoding for a dominant negative form of a MMR proteincan be introduced into the genome of an animal by producing a transgenicanimal. The animal can be any species for which suitable techniques areavailable to produce transgenic animals. For example, transgenic animalscan be prepared from domestic livestock, e.g., bovine, swine, sheep,goats, horses, etc.; from animals used for the production of recombinantproteins, e.g., bovine, swine, or goats that express a recombinantpolypeptide in their milk; or experimental animals for research orproduct testing, e.g., mice, rats, guinea pigs, hamsters, rabbits, etc.Cell lines that are determined to be MMR-defective can then be used as asource for producing genetically altered immunoglobulin genes in vitroby introducing whole, intact immunoglobulin genes and/or chimeric genesencoding for single chain antibodies into MMR-defective cells from anytissue of the MMR-defective animal.

Once a transfected cell line or a colony of transgenic animals has beenproduced, it can be used to generate new mutations in one or moregene(s) of interest. A gene of interest can be any gene naturallypossessed by the cell line or transgenic animal or introduced into thecell line or transgenic animal. An advantage of using such cells oranimals to induce mutations is that the cell or animal need not beexposed to mutagenic chemicals or radiation, which may have secondaryharmful effects, both on the object of the exposure and on the workers.However, chemical mutagens may be used in combination with MMRdeficiency, which renders such mutagens less toxic due to anundetermined mechanism. Hypermutable animals can then be bred andselected for those producing genetically variable B-cells that may beisolated and cloned to identify new cell lines that are useful forproducing genetically variable cells. Once a new trait is identified,the dominant negative MMR gene allele can be removed by directlyknocking out the allele by technologies used by those skilled in the artor by breeding to mates lacking the dominant negative allele to selectfor offspring with a desired trait and a stable genome. Anotheralternative is to use a CRE-LOX expression system, whereby the dominantnegative allele is spliced from the animal genome once an animalcontaining a genetically diverse immunoglobulin profile has beenestablished. Yet another alternative is the use of inducible vectorssuch as the steroid induced pIND (Invitrogen) or pMAM (Clonetech)vectors which express exogenous genes in the presence ofcorticosteroids.

Mutations can be detected by analyzing for alterations in the genotypeof the cells or animals, for example by examining the sequence ofgenomic DNA, cDNA, messenger RNA, or amino acids associated with thegene of interest. Mutations can also be detected by screening for theproduction of antibody titers. A mutant polypeptide can be detected byidentifying alterations in electrophoretic mobility, spectroscopicproperties, or other physical or structural characteristics of a proteinencoded by a mutant gene. One can also screen for altered function ofthe protein in situ, in isolated form, or in model systems. One canscreen for alteration of any property of the cell or animal associatedwith the function of the gene of interest, such as but not limited to Igsecretion.

Examples of nucleic acid sequences encoding mismatch repair proteinsuseful in the method of the invention include, but are not limited tothe following: PMS1 (SEQ ID NO: 1); PMS2 (SEQ ID NO:3); PMS2-134 (SEQ IDNO:5); PMSR2 (SEQ ID NO:7); PMSR3 (SEQ ID NO:9); MLH1 (SEQ ID NO:11);MLH3 (SEQ ID NO:13); MSH2 (SEQ ID NO:15); MSH3 (SEQ ID NO:17); MSH4 (SEQID NO:19); MSH5 (SEQ ID NO:21); MSH6 (SEQ ID NO:23); PMSR6 (SEQ IDNO:25); PMSL9 (SEQ ID NO:27); yeast MLH1 (SEQ ID NO:29); mouse PMS2 (SEQID NO:31); mouse PMS2-134 (SEQ ID NO:33); Arabidopsis thaliana PMS2 (SEQID NO:35); and Arabidopsis thaliana PMS2-134 (SEQ ID NO:37). Thecorresponding amino acid sequences for the listed nucleic acid sequencesare: PMS1 (SEQ ID NO:2); PMS2 (SEQ ID NO:4); PMS2-134 (SEQ ID NO:6);PMSR2 (SEQ ID NO:8); PMSR3 (SEQ ID NO:10); MLH1 (SEQ ID NO:12); MLH3(SEQ ID NO:14); MSH2 (SEQ ID NO:16); MSH3 (SEQ ID NO:18); MSH4 (SEQ IDNO:20); MSH5 (SEQ ID NO:22); MSH6 (SEQ ID NO:24); PMSR6 (SEQ ID NO:26);PMSL9 (SEQ ID NO:28); yeast MLH1 (SEQ ID NO:30); mouse PMS2 (SEQ IDNO:32); mouse PMS2-134 (SEQ ID NO:34); Arabidopsis thaliana PMS2 (SEQ IDNO:36); and Arabidopsis thaliana PMS2-134 (SEQ ID NO:38).

The invention also embraces in vitro immunization of cells that arecapable of producing antibodies such that the cells produceantigen-specific antibodies. The cells that are capable of producingantibodies are cells derived from sources containing lymphocytes such asthe peripheral blood, lymph nodes and spleen. Immunogens may includepurified antigens, denatured protein, solubilized cells, proteinmixtures, membrane preparations, whole cells, minced tissues and tumors,organisms, viruses, and the like. In the methods of the invention, theimmunogens may be conjugated with a mitogenic polypeptide, including,but not limited to at least a portion of tetanus toxoid, ovalbumin,bovine serum albumin, thyroglobulin, diptheria toxoid, BCG, keyholelimpet hemocyanin (KLH), and cholera toxin.

Antigens may be conjugated to mitogenic polypeptides in any way known inthe art. For example, fusion proteins may be generated by expressing apolypeptide in a recombinant expression system comprising thepolynucleotide encoding at least a portion of the antigen joinedin-frame to a polynucleotide encoding at least a portion of themitogenic polypeptide. The fusion protein may have the mitogenicpolypeptide joined at either the amino- or carboxy-terminus of theantigen. In some embodiments, more that one antigen may be expressed asa fusion protein in combination with a mitogenic polypeptide. In otherembodiments, more that one mitogenic polypeptide may be expressed as afusion protein with the antigen or antigens. In other embodiments, morethan one mitogenic polypeptide and more than one antigen may beexpressed together as a single fusion protein.

In an alternative embodiment, at least a portion of the mitogenicpolypeptide is conjugated to at least a portion of the antigen usingchemical cross-linkers. Examples of chemical cross-linkers include, butare not limited to gluteraldehyde, formaldehyde, 1,1-bis(diazoacetyl)-2-phenylethane, N-hydroxysuccinimide esters (e.g., esterswith 4-azidosalicylic acid, homobifunctional imidoesters includingdisuccinimidyl esters such as 3,3′-dithiobis (succinimidyl-propionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane).Derivatizing agents such asmethyl-3-[(p-azido-phenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, for example, a lysine residue in the mitogenicpolypeptide or antigen may be coupled to a C-terminal or other cysteineresidue in the antigen or mitogenic polypeptide, respectively, bytreatment with N-y-maleimidobutyryloxy-succinimide (Kitagawa and Aikawa(1976) J. Biochem. 79, 233-236).

Alternatively, a lysine residue in the mitogenic polypeptide or antigenmay be conjugated to a glutamic or aspartic acid residue in the antigenor mitogenic polypeptide, respectively, using isobutylchloroformate(Thorell and De Larson (1978) RADIOIMMUNOASSAY AND RELATED TECHNIQUES:METHODOLOGY AND CLINICAL APPLICATIONS, p. 288). Other coupling reactionsand reagents have been described in the literature

The conditions for the in vitro immunization procedure compriseincubating the cells at about 25-37° C., (preferably 37° C.) suppliedwith about 5-10% CO₂, in some embodiments, the incubation is performedwith between about 6-9% CO₂, in other embodiments the incubation isperformed in about 8% CO₂. The cell density is between about 2.5 to5×10⁶ cells/ml in culture medium. In some embodiments, the culturemedium is supplemented with about 2-20% FBS. In other embodiments, theculture medium is supplemented with about 5-15% FBS. In otherembodiments, the culture medium is supplemented with about 7-12% FBS. Inother embodiments, the culture medium is supplemented with about 10%FBS.

The in vitro stimulation culture medium is supplemented with cytokinesto stimulate the cells and increase the immune response. In general IL-2is supplied in the culture medium. However, other cytokines andadditives may also be included to increase the immune response. Suchcytokines and factors may include, for example, IL-4 and anti-CD40antibodies.

The immunogen-stimulated cells are fused to immortalized cells to createhybridoma cells. Typically, the immortalized cell is a myeloma cell. Thefusion of myeloma cells with the immunoglobulin-producing cells may beby any method known in the art for the creation of hybridoma cells.These methods include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110) (see also, Brown et al. (1981) J. Immunol. 127:539-546; Brownet al. (1980) J. Biol. Chem. 255 (11):4980-4983; Yeh et al. (1976) Proc.Natl. Acad. Sci. USA 76:2927-2931; and Yeh et al. (1982) Int. J. Cancer29:269-275), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al, 1985,MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). The hybridoma-producing the MAb of this invention may becultivated in vitro or in vivo.

The technology for producing monoclonal antibody hybridomas iswell-known to those of skill in the art and is described, for example inKenneth, in MONOCLONAL ANTIBODIES: A NEW DIMENSION IN BIOLOGICALANALYSES, Plenum Publishing Corp., New York, N.Y. (1980); Lerner (1981)Yale J. Biol. Med., 54:387-402; Galfre et al. (1977) Nature 266:55052;and Gefter et al. (1977) Somatic Cell Genet. 3:231-236. However, manyvariations of such methods are possible and would be appreciated by oneof skill in the art. Thus, the techniques for generation of hybridomasis not limited to the disclosures of these references.

Any myeloma cell may be used in the method of the invention. Preferably,the myeloma cells are human cells, but the invention is not limitedthereto or thereby. In some embodiments, the cells are sensitive tomedium containing hypoxanthine, aminopterin, and thymidine (HAT medium).In some embodiments, the myeloma cells do not express immunoglobulingenes. In some embodiments the myeloma cells are negative forEpstein-Barr virus (EBV) infection. In preferred embodiments, themyeloma cells are HAT-sensitive, EBV negative and Ig expressionnegative. Any suitable myeloma may be used. An example of such a myelomais that described in U.S. Pat. No. 4,720,459 to Winkelhake, anddeposited with the American Type Culture Collection (ATCC) as CRL 8644.Murine hybridomas may be generated using mouse myeloma cell lines (e.g.,the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines). Thesemurine myeloma lines are available from the ATCC.

The in vitro immunization procedure involves incubating the cells whichare capable of producing antibodies with an immunogen under conditionsthat promotes stimulation of the cells capable of producing antibodies.In some embodiments, the cells may be incubated in L-leucyl-L-lysinemethyl ester hydrobromide (LLOMe). While not wishing to be bound by anyparticular theory of operation, LLOme is believed to lysosomotropic andspecifically kills cytotoxic cells in the cell pool (such as NK cells,cytotoxic T cells, and CD8+ suppressor T cells) while not having aneffect on B cells, T helper cells accessory cells and fibroblasts(Borrebaeck (1988) Immunol Today 9(11):355-359). Generally, the cellsmay be incubated with LLOMe for a period of 1-30 minutes. In someembodiments, the incubation is performed for 10-20 minutes. In otherembodiments, the incubation is performed for 15 minutes. The LLOMe isgenerally a component of culture medium, such as, for example, RPMI1640, and is provided in a concentration of about 0.10 to 1 mM. In someembodiments, LLOMe is provided in an amount of about 0.10 to 0.50 mM. Inother embodiments, LLOMe is provided in an amount of about 0.25 mM.

In some embodiments of the method of the invention, the hybridoma cellsmay be rendered hypermutable by the introduction of a dominant negativeallele of a mismatch repair gene. The dominant negative allele of themismatch repair gene may be introduced into the hybridoma cell (i.e.,after the fusion of immunoglobulin-producing cells with the myelomacells) or may be introduced into the myeloma cell prior to the fusions.

The dominant negative allele of the mismatch repair gene is in the formof a polynucleotide which may be in the form of genomic DNA, cDNA, RNA,or a chemically synthesized polynucleotide. The polynucleotide can becloned into an expression vector containing a constitutively activepromoter segment (such as, but not limited to, CMV, SV40, EF-1 Dor LTRsequences) or to inducible promoter sequences such as those fromtetracycline, or ecdysone/glucocorticoid inducible vectors, where theexpression of the dominant negative mismatch repair gene can beregulated. The polynucleotide can be introduced into the cell bytransfection.

The hybridoma cells are screened for antibodies that specifically bindthe antigen used in the immunization procedure. In one embodiment, thecells are also screened for clones that express AID. These clones areexpected to have a higher rate of somatic hypermutation and class switchrecombination. These clones are selected and isolated to generateantibodies that specifically bind antigen and which perform CSR andsomatic hypermutation. Once a desired phenotype is achieved, one mayalso inactivate AID by any means known in the art, including but notlimited to knocking out all or part of the AID gene, by introducing aframeshift in the AID gene, by interrupting the AID gene with anothersequence by homologous recombination, and the like.

In other embodiments of the invention, the hybridoma cells are inducedto express AID by stimulating the hybridoma cells with activatingcytokines. The activating cytokines may be lipopolysaccharide (LPS),TGFβ, CD40L, IL-4 and combinations thereof.

In other embodiments of the invention, the hybridoma cells are inducedto express AID by transfecting the hybridoma cells with polynucleotidescomprising a sequence encoding AID operably linked to expression controlsequences. The hybridoma cells may constituitively express AID or beinduced to express AID. Once a desired phenotype is achieved, one caninactivate the AID by any means known in the art.

In other embodiments of the invention, in addition to selecting cellsthat express AID (either naturally or induced to express AID), the cellsmay be naturally defective in mismatch repair or be induced to bedefective in mismatch repair. The hybridoma cells may be defective inmismatch repair due to the fact that the cells that are capable ofproducing antibodies are naturally defective in mismatch repair.Alternatively, the immortalized cell may be naturally defective inmismatch repair. Alternatively, both the cells capable of producingantibodies and the immortalized cells may be naturally defective inmismatch repair. In some embodiments, the cells are manipulated to bedefective in mismatch repair due to knocking out one or more genesresponsible for mismatch repair, introducing a dominant negative alleleof a mismatch repair gene as described above, or by chemicallyinhibiting mismatch repair as described in Nicolaides et al., WO02/05456, “Chemical Inhibitors of Mismatch Repair,” the disclosure ofwhich is explicitly incorporated by reference herein in its entirety.

In another embodiment of the invention, the antibody-producing cells maybe hybridomas producing antibodies rather than hybridomas made de novo.In other embodiments, the antibody-producing cells may be mammalianexpression cells that produce antibodies due to transformation of thecells with polynucleotides encoding immunoglobulin heavy and lightchains. The expression cells may be expressing immunoglobulins orderivatives thereof. Such products include, for example, fully humanantibodies, human antibody homologs, humanized antibody homologs,chimeric antibody homologs, Fab, Fab′, F(ab′)₂ and F(v) antibodyfragments, single chain antibodies, and monomers or dimers of antibodyheavy or light chains or mixtures thereof. The known hybridomas andmammalian expression cells (as well as transfectomas) may be furthermanipulated as described above by inhibiting mismatch repair withsimulataneous or separate stimulation of expression of AID (or simpleselection of cells naturally expressing AID).

In each case, once a desired phenotype is achieved, genomic stabilitymay be restored as described above such that further mutation does notoccur.

The invention also comprises isolated antibody-producing cells producedby any of the foregoing methods.

For further information on the background of the invention the followingreferences may be consulted, each of which is incorporated herein byreference in its entirety:

-   1. Glaser, V. (1996) Can ReoPro repolish tarnished monoclonal    therapeutics? Nat. Biotechol. 14:1216-1217.-   2. Weiner, L. M. (1999) Monoclonal antibody therapy of cancer.    Semin. Oncol. 26:43-51.-   3. Saez-Llorens, X. E. et al. (1998) Safety and pharmacokinetics of    an intramuscular humanized monoclonal antibody to respiratory    syncytial virus in premature infants and infants with    bronchopulmonary dysplasia. Pediat. Infect. Dis. J. 17:787-791.-   4. Shield, C. F. et al. (1996) A cost-effective analysis of OKT3    induction therapy in cadaveric kidney transplantation. Am. J. Kidney    Dis. 27:855-864.-   5. Khazaeli, M. B. et al. (1994) Human immune response to monoclonal    antibodies. J. Immunother. 15:42-52.-   6. Emery, S. C. and W. J. Harris “Strategies for humanizing    antibodies” In: ANTIBODY ENGINEERING C. A. K. Borrebaeck (Ed.)    Oxford University Press, N.Y. 1995, pp. 159-183.-   7. U.S. Pat. No. 5,530,101 to Queen and Selick.-   8. Reff, M. E. (1993) High-level production of recombinant    immunoglobulins in mammalian cells. Curr. Opin. Biotechnol.    4:573-576.-   9. Neuberger, M. and M. Gruggermann, (1997) Monoclonal antibodies.    Mice perform a human repertoire. Nature 386:25-26.-   10. Fiedler, U. and U. Conrad (1995) High-level production and    long-term storage of engineered antibodies in transgenic tobacco    seeds. Bio/Technology 13:1090-1093.-   11. Baker S. M. et al. (1995) Male defective in the DNA mismatch    repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis.    Cell 82:309-319.-   12. Bronner, C. E. et al. (1994) Mutation in the DNA mismatch repair    gene homologue hMLH1 is associated with hereditary non-polyposis    colon cancer. Nature 368:258-261.-   13. de Wind N. et al. (1995) Inactivation of the mouse Msh2 gene    results in mismatch repair deficiency, methylation tolerance,    hyperrecombination, and predisposition to cancer. Cell 82:321-300.-   14. Drummond, J. T. et al. (1995) Isolation of an hMSH2-p160    heterodimer that restores mismatch repair to tumor cells. Science    268:1909-1912.-   15. Modrich, P. (1994) Mismatch repair, genetic stability, and    cancer. Science 266: 1959-1960.-   16. Nicolaides, N. C. et al. (1998) A Naturally Occurring hPMS2    Mutation Can Confer a Dominant Negative Mutator Phenotype. Mol.    Cell. Biol. 18:1635-1641.-   17. Prolla, T. A. et al. (1994) MLH1, PMS1, and MSH2 Interaction    during the initiation of DNA mismatch repair in yeast. Science    264:1091-1093.-   18. Strand, M. et al. (1993) Destabilization of tracts of simple    repetitive DNA in yeast by mutations affecting DNA mismatch repair.    Nature 365 :274-276.-   19. Su, S. S., R. S. Lahue, K. G. Au, and P. Modrich (1988) Mispair    specificity of methyl directed DNA mismatch corrections in vitro. J.    Biol. Chem. 263:6829-6835.-   20. Parsons, R. et al. (1993) Hypermutability and mismatch repair    deficiency in RER+ tumor cells. Cell 75:1227-1236.-   21. Papadopoulos, N. et al. (1993) Mutation of a mutL homolog is    associated with hereditary colon cancer. Science 263:1625-1629.-   22. Perucho, M. (1996) Cancer of the microsatellite mutator    phenotype. Biol. Chem. 377:675-684.-   23. Nicolaides N. C., K. W. Kinzler, and B. Vogelstein (1995)    Analysis of the 5′ region of PMS2 reveals heterogenous transcripts    and a novel overlapping gene. Genomics 29:329-334.-   24. Nicolaides, N. C. et al. (1995) Genomic organization of the    human PMS2 gene family. Genomics 30:195-206.-   25. Palombo, F. et al. (1994) Mismatch repair and cancer. Nature    36:417.-   26. Eshleman J. R. and S. D. Markowitz (1996) Mismatch repair    defects in human carcinogenesis. Hum. Mol. Genet. 5:1489-494.-   27. Liu, T. et al. (2000) Microsatellite instability as a predictor    of a mutation in a DNA mismatch repair gene in familial colorectal    cancer. Genes Chromosomes Cancer 27:17-25.-   28. Nicolaides, N. C. et al. (1992) The Jun family members, c-JUN    and JUND, transactivate the human c-myb promoter via an Ap1 like    element. J. Biol. Chem. 267: 19665-19672.-   29. Shields, R. L. et al (1995) Anti-IgE monoclonal antibodies that    inhibit allergen-specific histamine release. Int. Arch. Allergy    Immunol. 107:412-413.-   30. Frigerio L. et al. (2000) Assembly, secretion, and vacuolar    delivery of a hybrid immunoglobulin in plants. Plant Physiol.    123:1483-1494.-   31. Bignami M, (2000) Unmasking a killer: DNA O(6)-methylguanine and    the cytotoxicity of methylating agents. Mutat. Res. 462:71-82.-   32. Drummond, J. T. et al. (1996) Cisplatin and adriamycin    resistance are associated with MutLa and mismatch repair deficiency    in an ovarian tumor cell line. J. Biol. Chem. 271:9645-19648.-   33. Galio, L. et al (1999) ATP hydrolysis-dependent formation of a    dynamic ternary nucleoprotein complex with MutS and MutL. Nucl.    Acids Res. 27:2325-23231.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLES Example 1 Stable Expression of Dominant Negative MMR Genes inHybridoma Cells

It has been previously shown by Nicolaides et al. (Nicolaides et al.(1998) A Naturally Occurring hPMS2 Mutation Can Confer a DominantNegative Mutator Phenotype Mol. Cell. Biol. 18:1635-1641) that theexpression of a dominant negative allele in an otherwise MMR-proficientcell could render these host cells MMR deficient. The creation ofMMR-deficient cells can lead to the generation of genetic alterationsthroughout the entire genome of a host organisms offspring, yielding apopulation of genetically altered offspring or siblings that may producebiochemicals with altered properties. This patent application teaches ofthe use of dominant negative MMR genes in antibody-producing cells,including but not limited to rodent hybridomas, human hybridomas,chimeric rodent cells producing human immunoglobulin gene products,human cells expressing immunoglobulin genes, mammalian cells producingsingle chain antibodies, and prokaryotic cells producing mammalianimmunoglobulin genes or chimeric immunoglobulin molecules such as thosecontained within single-chain antibodies. The cell expression systemsdescribed above that are used to produce antibodies are well known bythose skilled in the art of antibody therapeutics.

To demonstrate the ability to create MMR defective hybridomas usingdominant negative alleles of MMR genes, we first transfected a mousehybridoma cell line that is known to produce an antibody directedagainst the human IgE protein with an expression vector containing thehuman PMS2 (cell line referred to as HBPMS2), the previously publisheddominant negative PMS2 mutant referred herein as PMS134 (cell linereferred to as HB134), or with no insert (cell line referred to asHBvec). The results showed that the PMS134 mutant could indeed exert arobust dominant negative effect, resulting in biochemical and geneticmanifestations of MMR deficiency. Unexpectedly it was found that thefull length PMS2 also resulted in a lower MMR activity while no effectwas seen in cells containing the empty vector. A brief description ofthe methods is provided below.

The MMR-proficient mouse H36 hybridoma cell line was transfected withvarious hPMS2 expression plasmids plus reporter constructs for assessingMMR activity. The MMR genes were cloned into the pEF expression vector,which contains the elongation factor promoter upstream of the cloningsite followed by a mammalian polyadenylation signal. This vector alsocontains the NEOr gene that allows for selection of cells retaining thisplasmid. Briefly, cells were transfected with 1 μg of each vector usingpolyliposomes following the manufacturer's protocol (Life Technologies).Cells were then selected in 0.5 mg/ml of G418 for 10 days and G418resistant cells were pooled together to analyze for gene expression. ThepEF construct contains an intron that separates the exon 1 of the EFgene from exon 2, which is juxtaposed to the 5′ end of the polylinkercloning site. This allows for a rapid reverse transcriptase polymerasechain reaction (RT-PCR) screen for cells expressing the splicedproducts. At day 17, 100,000 cells were isolated and their RNA extractedusing the trizol method as previously described (Nicolaides N. C.,Kinzler, K. W., and Vogelstein, B. (1995) Analysis of the 5′ region ofPMS2 reveals heterogeneous transcripts and a novel overlapping gene.Genomics 29:329-334). RNAs were reverse-transcribed using Superscript II(Life Technologies) and PCR-amplified using a sense primer located inexon 1 of the EF gene (5′-ttt cgc aac ggg ttt gcc g-3′) (SEQ ID NO:49)and an antisense primer (5′-gtt tca gag tta agc ctt cg-3′) (SEQ IDNO:50) centered at nt 283 of the published human PMS2 cDNA, which willdetect both the full length as well as the PMS134 gene expression.Reactions were carried out using buffers and conditions as previouslydescribed (Nicolaides, N. C., et al. (1995) Genomic organization of thehuman PMS2 gene family. Genomics 30:195-206), using the followingamplification parameters: 94° C. for 30 sec, 52° C. for 2 min, 72° C.for 2 min, for 30 cycles. Reactions were analyzed on agarose gels. FIG.1 shows a representative example of PMS expression in stably transducedH36 cells.

Expression of the protein encoded by these genes were confirmed viawestern blot using a polyclonal antibody directed to the first 20 aminoacids located in the N-terminus of the protein following the procedurespreviously described (data not shown) (Nicolaides et al. (1998) ANaturally Occurring hPMS2 Mutation Can Confer a Dominant NegativeMutator Phenotype. Mol. Cell. Biol. 18:1635-1641).

Example 2 hPMS134 Causes a Defect in MMR Activity and Hypermutability inHybridoma Cells

A hallmark of MMR deficiency is the generation of unstablemicrosatellite repeats in the genome of host cells. This phenotype isreferred to as microsatellite instability (MI) (Modrich, P. (1994)Mismatch repair, genetic stability, and cancer Science 266:1959-1960;Palombo, F., et al. (1994) Mismatch repair and cancer Nature 36:417). MIconsists of deletions and/or insertions within repetitive mono-, di-and/or tri-nucleotide repetitive sequences throughout the entire genomeof a host cell. Extensive genetic analysis eukaryotic cells have foundthat the only biochemical defect that is capable of producing MI isdefective MMR (Strand, M., et al. (1993) Destabilization of tracts ofsimple repetitive DNA in yeast by mutations affecting DNA mismatchrepair Nature 365:274-276; Perucho, M. (1996) Cancer of themicrosatellite mutator phenotype. Biol Chem. 377:675-684; Eshleman J.R., and Markowitz, S. D. (1996) Mismatch repair defects in humancarcinogenesis. Hum. Mol. Genet. 5:1489-494). In light of this uniquefeature that defective MMR has on promoting MI, it is now used as abiochemical marker to survey for lack of MMR activity within host cells(Perucho, M. (1996) Cancer of the microsatellite mutator phenotype.Biol. Chem. 377:675-684; Eshleman J. R., and Markowitz, S. D. (1996)Mismatch repair defects in human carcinogenesis. Hum. Mol. Genet.5:1489-494; Liu, T., et al. (2000) Microsatellite instability as apredictor of a mutation in a DNA mismatch repair gene in familialcolorectal cancer Genes Chromosomes Cancer 27:17-25).

A method used to detect MMR deficiency in eukaryotic cells is to employa reporter gene that has a polynucleotide repeat inserted within thecoding region that disrupts its reading frame due to a frame shift. Inthe case where MMR is defective, the reporter gene will acquire randommutations (i.e. insertions and/or deletions) within the polynucleotiderepeat yielding clones that contain a reporter with an open readingframe. We have employed the use of an MMR-sensitive reporter gene tomeasure for MMR activity in HBvec, HBPMS2, and HBPMS134 cells. Thereporter construct used the pCAR-OF, which contains a hygromycinresistance (HYG) gene plus a β-galactosidase gene containing a 29 bpout-of-frame poly-CA tract at the 5′ end of its coding region. ThepCAR-OF reporter would not generate β-galactosidase activity unless aframe-restoring mutation (i.e., insertion or deletion) arose followingtransfection. HBvec, HBPMS2, and HB134 cells were each transfected withpCAR-OF vector in duplicate reactions following the protocol describedin Example 1. Cells were selected in 0.5 mg/ml G418 and 0.5 mg/ml HYG toselect for cells retaining both the MMR effector and the pCAR-OFreporter plasmids. All cultures transfected with the pCAR vectorresulted in a similar number of HYG/G418 resistant cells. Cultures werethen expanded and tested for β-galactosidase activity in situ as well asby biochemical analysis of cell extracts. For in situ analysis, 100,000cells were harvested and fixed in 1% gluteraldehyde, washed in phosphatebuffered saline solution and incubated in 1 ml of X-gal substratesolution [0.15 M NaCl, 1 mM MgCl₂, 3.3 mM K₄Fe(CN)₆, 3.3 mM K₃Fe(CN)₆,0.2% X-Gal] in 24 well plates for 2 hours at 37° C. Reactions werestopped in 500 mM sodium bicarbonate solution and transferred tomicroscope slides for analysis. Three fields of 200 cells each werecounted for blue (β-galactosidase positive cells) or white(β-galactosidase negative cells) to assess for MMR inactivation. Table 1shows the results from these studies. While no β-galactosidase positivecells were observed in HBvec cells, 10% of the cells per field wereβ-galactosidase positive in HB134 cultures and 2% of the cells per fieldwere β-galactosidase positive in HBPMS2 cultures.

Cell extracts were prepared from the above cultures to measureβ-galactosidase using a quantitative biochemical assay as previouslydescribed (Nicolaides et al. (1998) A Naturally Occurring hPMS2 MutationCan Confer a Dominant Negative Mutator Phenotype Mol. Cell. Biol.18:1635-1641; Nicolaides, N. C., et al. (1992) The Jun family members,c-JUN and JUND, transactivate the human c-myb promoter via an Ap1 likeelement. J. Biol. Chem. 267:19665-19672). Briefly, 100,000 cells werecollected, centrifuged and resuspended in 200 μls of 0.25M Tris, pH 8.0.Cells were lysed by freeze/thawing three times and supernatantscollected after microfugation at 14,000 rpms to remove cell debris.Protein content was determined by spectrophotometric analysis at OD²⁸⁰.For biochemical assays, 20 μg of protein was added to buffer containing45 mM 2-mercaptoethanol, 1 mM MgCl₂, 0.1 M NaPO₄ and 0.6 mg/mlChlorophenol red-β-D-galactopyranoside (CPRG, Boehringer Mannheim).Reactions were incubated for 1 hour, terminated by the addition of 0.5 MNa₂CO₃, and analyzed by spectrophotometry at 576 nm. H36 cell lysateswere used to subtract out background. FIG. 2 shows the β-galactosidaseactivity in extracts from the various cell lines. As shown, the HB134cells produced the highest amount of β-galactosidase, while no activitywas found in the HBvec cells containing the pCAR-OF. These datademonstrate the ability to generate MMR defective hybridoma cells usingdominant negative MMR gene alleles.

TABLE 1 Table 1. β-galactosidase expression of HBvec, HBPMS2 and HB134cells transfected with pCAR-OF reporter vectors. Cells were transfectedwith the pCAR-OF β-galactosidase reporter plasmid. Transfected cellswere selected in hygromycin and G418, expanded and stained with X-galsolution to measure for β-galactosidase activity (blue colored cells). 3fields of 200 cells each were analyzed by microscopy. The results belowrepresent the mean +/− standard deviation of these experiments. CELLLINE # BLUE CELLS HBvec 0 +/− 0 HBPMS2 4 +/− 1 HB134 20 +/− 3 

Example 3 Screening Strategy to Identify Hybridoma Clones ProducingAntibodies with Higher Binding Affinities and/or IncreasedImmunoglobulin Production

An application of the methods presented within this document is the useof MMR deficient hybridomas or other immunoglobulin producing cells tocreate genetic alterations within an immunoglobulin gene that will yieldantibodies with altered biochemical properties. An illustration of thisapplication is demonstrated within this example whereby the HB134hybridoma (see Example 1), which is a MMR-defective cell line thatproduces an anti-human immunoglobulin type E (hIge) MAb, is grown for 20generations and clones are isolated in 96-well plates and screened forhIgE binding. FIG. 3 outlines the screening procedure to identify clonesthat produce high affinity MAbs, which is presumed to be due to analteration within the light or heavy chain variable region of theprotein. The assay employs the use of a plate Enzyme LinkedImmunosorbant Assay (ELISA) to screen for clones that producehigh-affinity MAbs. 96-well plates containing single cells from HBvec orHB134 pools are grown for 9 days in growth medium (RPMI 1640 plus 10%fetal bovine serum) plus 0.5 mg/ml G418 to ensure clones retain theexpression vector. After 9 days, plates are screened using an hIgE plateELISA, whereby a 96 well plate is coated with 50 μls of a 1 μg/ml hIgEsolution for 4 hours at 4° C. Plates are washed 3 times in calcium andmagnesium free phosphate buffered saline solution (PBS−/−) and blockedin 100 μls of PBS^(−/−) with 5% dry milk for 1 hour at room temperature.Wells are rinsed and incubated with 100 μls of a PBS solution containinga 1:5 dilution of conditioned medium from each cell clone for 2 hours.Plates are then washed 3 times with PBS−/− and incubated for 1 hour atroom temperature with 50 μls of a PBS^(−/−) solution containing 1:3000dilution of a sheep anti-mouse horse radish peroxidase (HRP) conjugatedsecondary antibody. Plates are then washed 3 times with PBS−/− andincubated with 50 μls of TMB-HRP substrate (BioRad) for 15 minutes atroom temperature to detect amount of antibody produced by each clone.Reactions are stopped by adding 50 pls of 500 mM sodium bicarbonate andanalyzed by OD at 415 nm using a BioRad plate reader. Clones exhibitingan enhanced signal over background cells (H36 control cells) are thenisolated and expanded into 10 ml cultures for additionalcharacterization and confirmation of ELISA data in triplicateexperiments. ELISAs are also performed on conditioned (CM) from the sameclones to measure total Ig production within the conditioned medium ofeach well. Clones that produce an increased ELISA signal and haveincreased antibody levels are then further analyzed for variants thatover-express and/or over-secrete antibodies as described in Example 4.Analysis of five 96-well plates each from HBvec or HB134 cells havefound that a significant number of clones with a higher Optical Density(OD) value is observed in the MMR-defective HB134 cells as compared tothe HBvec controls. FIG. 4 shows a representative example of HB134clones producing antibodies that bind to specific antigen (in this caseIgE) with a higher affinity. FIG. 4 provides raw data from the analysisof 96 wells of HBvec (left graph) or HB134 (right graph) which shows 2clones from the HB134 plate to have a higher OD reading due to 1)genetic alteration of the antibody variable domain that leads to anincreased binding to IgE antigen, or 2) genetic alteration of a cellhost that leads to over-production/secretion of the antibody molecule.Anti-Ig ELISA found that the two clones shown in FIG. 4 have Ig levelswithin their CM similar to the surrounding wells exhibiting ower ODvalues. These data suggest that a genetic alteration occurred within theantigen binding domain of the antibody which in turn allows for higherbinding to antigen.

Clones that produced higher OD values as determined by ELISA werefurther analyzed at the genetic level to confirm that mutations withinthe light or heavy chain variable region have occurred that lead to ahigher binding affinity hence yielding to a stronger ELISA signal.Briefly, 100,000 cells are harvested and extracted for RNA using theTriazol method as described above. RNAs are reverse transcribed usingSuperscript II as suggested by the manufacturer (Life Technology) andPCR-amplified for the antigen binding sites contained within thevariable light and heavy chains. Because of the heterogeneous nature ofthese genes, the following degenerate primers are used to amplify lightand heavy chain alleles from the parent H36 strain.

Light chain sense: (SEQ ID NO: 45) 5′-GGA TTT TCA GGT GCA GAT TTT CAG-3′Light chain antisense: (SEQ ID NO: 46) 5′-ACT GGA TGG TGG GAA GAT GGA-3′Heavy chain sense: (SEQ ID NO: 47) 5′-A(G/T) GTN (A/C)AG CTN CAG (C/G)AGTC-3′ Heavy chain antisense: (SEQ ID NO: 48) 5′-TNC CTT G(A/G)C CCC AGTA(G/A)(A/T)C-3′

PCR reactions using degenerate oligonucleotides are carried out at 94°C. for 30 sec, 52° C. for 1 min, and 72° C. for 1 min for 35 cycles.Products are analyzed on agarose gels. Products of the expectedmolecular weights are purified from the gels by Gene Clean (Bio 101),cloned into T-tailed vectors, and sequenced to identify the wild typesequence of the variable light and heavy chains. Once the wild typesequence has been determined, non-degenerate primers were made forRT-PCR amplification of positive HB134 clones. Both the light and heavychains were amplified, gel purified and sequenced using thecorresponding sense and antisense primers. The sequencing of RT-PCRproducts gives representative sequence data of the endogenousimmunoglobulin gene and not due to PCR-induced mutations. Sequences fromclones were then compared to the wild type sequence for sequencecomparison. An example of the ability to create in vivo mutations withinan immunoglobulin light or heavy chain is shown in FIG. 5, where HB134clone92 was identified by ELISA to have an increased signal for hIgE.The light chain was amplified using specific sense and antisenseprimers. The light chain was RT-PCR amplified and the resulting productwas purified and analyzed on an automated ABI377 sequencer. As shown inclone A, a residue −4 upstream of the CDR region 3 had a genetic changefrom ACT to TCT, which results in a Thr to Ser change within theframework region just preceding the CDR#3. In clone B, a residue −6upstream of the CDR region had a genetic change from CCC to CTC, whichresults in a Pro to Leu change within framework region preceding CDR#2.

The ability to generate random mutations in immunoglobulin genes orchimeric immunoglobulin genes is not limited to hybridomas. Nicolaideset al. ((1998) A Naturally Occurring hPMS2 Mutation Can Confer aDominant Negative Mutator Phenotype Mol. Cell. Biol. 18:1635-1641) haspreviously shown the ability to generate hypermutable hamster cells andproduce mutations within an endogenous gene. A common method forproducing humanized antibodies is to graft CDR sequences from a MAb(produced by immunizing a rodent host) onto a human Ig backbone andtransfect the chimeric genes into Chinese Hamster Ovary (CHO) cellswhich in turn produce a functional Ab that is secreted by the CHO cells(Shields, R. L., et al. (1995) Anti-IgE monoclonal antibodies thatinhibit allergen-specific histamine release. Int. Arch. Allergy Immunol.107:412-413). The methods described within this application are alsouseful for generating genetic alterations within Ig genes or chimericIgs transfected within host cells such as rodent cell lines, plants,yeast and prokaryotes (Frigerio L, et al. (2000) Assembly, secretion,and vacuolar delivery of a hybrid immunoglobulin in plants. PlantPhysiol. 123:1483-1494).

These data demonstrate the ability to generate hypermutable hybridomas,or other Ig producing host cells that can be grown and selected, toidentify structurally altered immunoglobulins yielding antibodies withenhanced biochemical properties, including but not limited to increasedantigen binding affinity. Moreover, hypermutable clones that containmissense mutations within the immunoglobulin gene that result in anamino acid change or changes can be then further characterized for invivo stability, antigen clearance, on-off binding to antigens, etc.Clones can also be further expanded for subsequent rounds of in vivomutations and can be screened using the strategy listed above.

The use of chemical mutagens to produce genetic mutations in cells orwhole organisms are limited due to the toxic effects that these agentshave on “normal” cells. The use of chemical mutagens such as MNU in MMRdefective organisms is much more tolerable yielding to a 10 to 100 foldincrease in genetic mutation over MMR deficiency alone (Bignami M,(2000) Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicityof methylating agents. Mutat. Res. 462:71-82). This strategy allows forthe use of chemical mutagens to be used in MMR-defective Ab producingcells as a method for increasing additional mutations withinimmunoglobulin genes or chimeras that may yield functional Abs withaltered biochemical properties such as enhanced binding affinity toantigen, etc.

Example 4 Generation of Antibody Producing Cells with Enhanced AntibodyProduction

Analysis of clones from H36 and HB134 following the screening strategylisted above hasidentified a significant number of clones that produceenhanced amounts of antibody into the medium. While a subset of theseclones gave higher Ig binding data as determined by ELISA as aconsequence of mutations within the antigen binding domains contained inthe variable regions, others were found to contain “enhanced” antibodyproduction. A summary of the clones producing enhanced amounts ofsecreted MAb is shown in TABLE 2, where a significant number of clonesfrom HB134 cells were found to produce enhanced Ab production within theconditioned medium as compared to H36 control cells.

TABLE 2. Generation of hybridoma cells producing high levels ofantibody. HB134 clones were assayed by ELISA for elevated Ig levels.Analysis of 480 clones showed that a significant number of clones hadelevated MAb product levels in their CM. Quantification showed thatseveral of these clones produced greater than 500 ngs/ml of MAb due toeither enhanced expression and/or secretion as compared to clones fromthe H36 cell line.

TABLE 2 Production of MAb in CM from H36 and HB134 clones. Cell Line %clones > 400 ng/ml % clones > 500 ng/ml H36  1/480 = 0.2% 0/480 = 0%HB134 50/480 = 10% 8/480 = 1.7%

Cellular analysis of HB134 clones with higher MAb levels within theconditioned medium (CM) were analyzed to determine if the increasedproduction was simply due to genetic alterations at the Ig locus thatmay lead to over-expression of the polypeptides forming the antibody, ordue to enhanced secretion due to a genetic alteration affectingsecretory pathway mechanisms. To address this issue, we expanded threeHB134 clones that had increased levels of antibody within their CM.10,000 cells were prepared for western blot analysis to assay forintracellular steady state Ig protein levels (FIG. 6). In addition, H36cells were used as a standard reference (Lane 2) and a rodent fibroblast(Lane 1) was used as an Ig negative control. Briefly, cells werepelleted by centrifugation and lysed directly in 300 μl of SDS lysisbuffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M2-mercaptoethanol, 0.001% bromophenol blue) and boiled for 5 minutes.Lysate proteins were separated by electrophoresis on 4-12% NuPAGE gels(for analysis of Ig heavy chain. Gels were electroblotted ontoImmobilon-P (Millipore) in 48 mM Tris base, 40 mM glycine, 0.0375% SDS,20% methanol and blocked at room temperature for 1 hour in Tris-bufferedsaline (TBS) plus 0.05% Tween-20 and 5% condensed milk. Filters wereprobed with a 1:10,000 dilution of sheep anti-mouse horseradishperoxidase conjugated monoclonal antibody in TBS buffer and detected bychemiluminescence using Supersignal substrate (Pierce). Experiments wererepeated in duplicates to ensure reproducibility. FIG. 6 shows arepresentative analysis where a subset of clones had enhanced Igproduction which accounted for increased Ab production (Lane 5) whileothers had a similar steady state level as the control sample, yet hadhigher levels of Ab within the CM. These data suggest a mechanismwhereby a subset of HB134 clones contained a genetic alteration that inturn produces elevated secretion of antibody.

The use of chemical mutagens to produce genetic mutations in cells orwhole organisms are limited due to the toxic effects that these agentshave on “normal” cells. The use of chemical mutagens such as MNU in MMRdefective organisms is much more tolerable yielding to a 10 to 100 foldincrease in genetic mutation over MMR deficiency alone (Bignami M,(2000) Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicityof methylating agents. Mutat. Res. 462:71-82). This strategy allows forthe use of chemical mutagens to be used in MMR-defective Ab-producingcells as a method for increasing additional mutations withinimmunoglobulin genes or chimeras that may yield functional Abs withaltered biochemical properties such as enhanced binding affinity toantigen, etc.

Example 5 Establishment of Genetic Stability in Hybridoma Cells with NewOutput Trait

The initial steps of MMR are dependent on two protein complexes, calledMutSα and MutLα (Nicolaides et al. (1998) A Naturally Occurring hPMS2Mutation Can Confer a Dominant Negative Mutator Phenotype. Mol. Cell.Biol. 18:1635-1641). Dominant negative MMR alleles are able to perturbthe formation of these complexes with downstream biochemicals involvedin the excision and polymerization of nucleotides comprising the“corrected” nucleotides. Examples from this application show the abilityof a truncated MMR allele (PMS134) as well as a full length human PMS2when expressed in a hybridoma cell line is capable of blocking MMRresulting in a hypermutable cell line that gains genetic alterationsthroughout its entire genome per cell division. Once a cell line isproduced that contains genetic alterations within genes encoding for anantibody, a single chain antibody, over-expression of immunoglobulingenes and/or enhanced secretion of antibody, it is desirable to restorethe genomic integrity of the cell host. This can be achieved by the useof inducible vectors whereby dominant negative MMR genes are cloned intosuch vectors, introduced into Ab-producing cells and the cells arecultured in the presence of inducer molecules and/or conditions.Inducible vectors include but are not limited to chemical regulatedpromoters such as the steroid inducible MMTV, tetracycline regulatedpromoters, temperature sensitive MMR gene alleles, and temperaturesensitive promoters.

The results described above lead to several conclusions. First,expression of hPMS2 and PMS2-134 results in an increase inmicrosatellite instability in hybridoma cells. That this elevatedmicrosatellite instability is due to MMR deficiency was proven byevaluation of extracts from stably transduced cells. The expression ofPMS2-134 results in a polar defect in MMR, which was only observed usingheteroduplexes designed to test repair from the 5′ direction (nosignificant defect in repair from the 3′ direction was observed in thesame extracts) (Nicolaides et al (1998) A Naturally Occurring hPMS2Mutation Can Confer a Dominant Negative Mutator Phenotype. Mol. Cell.Biol. 18:1635-1641). Interestingly, cells deficient in hMLH1 also have apolar defect in MMR, but in this case preferentially affecting repairfrom the 3′ direction (Drummond, J. T, et al (1996) Cisplatin andadriamycin resistance are associated with MutLa and mismatch repairdeficiency in an ovarian tumor cell line. J. Biol. Chem.271:9645-19648). It is known from previous studies in both prokaryotesand eukaryotes that the separate enzymatic components mediate repairfrom the two different directions. Our results, in combination withthose of Drummond et al. (Shields, R. L., et al (1995) Anti-IgEmonoclonal antibodies that inhibit allergen-specific histamine release.Int. Arch Allergy Immunol. 107:412-413), strongly suggest a model inwhich 5′ repair is primarily dependent on hPMS2 while 3′ repair isprimarily dependent on hMLH1. It is easy to envision how the dimericcomplex between PMS2 and MLH1 might set up this directionality. Thecombined results also demonstrate that a defect in directional MMR issufficient to produce a MMR-defective phenotype and suggests that anyMMR gene allele is useful to produce genetically altered hybridomacells, or a cell line that is producing Ig gene products. Moreover, theuse of such MMR alleles will be useful for generating geneticallyaltered Ig polypeptides with altered biochemical properties as well ascell hosts that produce enhanced amounts of antibody molecules.

Another method that is taught in this application is that any methodused to block MMR can be performed to generate hypermutablility in anantibody-producing cell that can lead to genetically altered antibodieswith enhanced biochemical features such as but not limited to increasedantigen binding, enhanced pharmacokinetic profiles, etc. These processescan also to be used to generate antibody producer cells that haveincreased Ig expression as shown in Example 4, FIG. 6 and/or increasedantibody secretion as shown in Table 2.

In addition, we demonstrate the utility of blocking MMR inantibody-producing cells to increase genetic alterations within Ig genesthat may lead to altered biochemical features such as, but not limitedto, increased antigen-binding affinities (FIGS. 5A and 5B). The blockadeof MMR in such cells can be through the use of dominant negative MMRgene alleles from any species including bacteria, yeast, protozoa,insects, rodents, primates, mammalian cells, and man. Blockade of MMRcan also be generated through the use of antisense RNA ordeoxynucleotides directed to any of the genes involved in the MMRbiochemical pathway. Blockade of MMR can be through the use ofpolypeptides that interfere with subunits of the MMR complex includingbut not limited to antibodies. Finally, the blockade of MMR may bethrough the use chemicals such as but not limited to nonhydrolyzable ATPanalogs, which have been shown to block MMR (Galio, L, et al. (1999) ATPhydrolysis-dependent formation of a dynamic ternary nucleoproteincomplex with MutS and MutL. Nucl. Acids Res. 27:2325-23231).

Example 6

To demonstrate that cells may be selected that express AID, cDNA fromhybridoma cells generated by in vitro immunization was generated usingeither SuperScript II (for clones 5-8) or ExpressDirect (for clones 7-6,8-2 and 3-32). AID (Genbank Accession No.: NM_(—)020661 (SEQ ID NO:39)which encodes the AID protein (SEQ ID NO:40)) was amplified usingprimers AID-77-F (ATGGACAGCCTCTTGATGAA) (SEQ ID NO:41) andAID-561-R(CAGGCTTTGAAAGTTCTTTC) (SEQ ID NO:42) to generate an ampliconof 484 bp. PCR conditions: 95° C., 5 min. 1×; 94° C., 30 sec; 55° C., 30sec; 72° C., 30 sec 35×; 72° C., 7 min. 1×. 10% of reaction mixture wasanalysed on a 1% agarose gel. The results are shown in FIG. 7.

1. An isolated, hypermutable, antibody-producing cell produced in vitro by introducing into the antibody-producing cell a polynucleotide comprising a nucleic acid sequence encoding a PMS2 having an ATPase domain, wherein expression of said polynucleotide inhibits mismatch repair, wherein inhibition of mismatch repair stimulates expression of activation-induced cytidine deaminase, thereby generating a hypermutable antibody-producing cell.
 2. The cell of claim 1, wherein said polynucleotide encodes a mammalian PMS2 having an ATPase domain.
 3. The cell of claim 1, wherein said polynucleotide encodes a rodent PMS2 having an ATPase domain.
 4. The cell of claim 1, wherein said polynucleotide encodes a human PMS2 having an ATPase domain.
 5. The cell of claim 1, wherein said polynucleotide encodes a plant PMS2 having an ATPase domain.
 6. The cell of claim 1 wherein said PMS2 is PMS2-134.
 7. The cell of claim 6 wherein said PMS2-134 is encoded by the nucleic acid sequence SEQ ID NO:5, SEQ ID NO:33, or SEQ ID NO:37.
 8. A cell culture comprising the cell of claim
 1. 9. The cell of claim 1, wherein the polynucleotide is inactivated, thereby producing a genetically stable antibody-producing cell.
 10. A cell culture comprising the cell of claim
 9. 11. A hybridoma cell producing antibodies produced from in vitro immunized immunoglobulin-producing cells by: (a) combining cells capable of producing immunoglobulins with an immunogenic antigen in vitro; (b) fusing said cells with myeloma cells to form parental hybridoma cells, wherein a polynucleotide comprising a nucleic acid sequence encoding a PMS2 having an ATPase domain is transfected into either said cells capable of producing immunoglobulins, said myeloma cells, or said parental hybridoma cells, wherein expression of said polynucleotide inhibits mismatch repair; (c) selecting for mismatch repair-inhibited hybridoma cells exhibiting increased expression of activation-induced cytidine deaminase relative to mismatch repair-proficient hybridoma cells, wherein expression of activation-induced cytidine deaminase is stimulated by said inhibition of mismatch repair; (d) incubating the selected mismatch repair-inhibited hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies that specifically bind antigen, thereby producing hybridoma cells producing antibodies from in vitro immunized immunoglobulin-producing cells.
 12. The hybridoma cell of claim 11, wherein said activation-induced cytidine deaminase is a mammalian activation-induced cytidine deaminase.
 13. The hybridoma cell of claim 11, wherein said activation-induced cytidine deaminase is a human activation-induced cytidine deaminase.
 14. The hybridoma cell of claim 11, wherein said activation-induced cytidine deaminase is a mouse activation-induced cytidine deaminase.
 15. The cell of claim 11, wherein said polynucleotide encodes a mammalian PMS2 having an ATPase domain.
 16. The cell of claim 11, wherein said polynucleotide encodes a rodent PMS2 having an ATPase domain.
 17. The cell of claim 11, wherein said polynucleotide encodes a human PMS2 having an ATPase domain.
 18. The cell of claim 11, wherein said polynucleotide encodes a plant PMS2 having an ATPase domain.
 19. The hybridoma cell of claim 11, wherein said PMS2 is PMS2-134.
 20. The hybridoma cell of claim 19, wherein said PMS2-134 is encoded by the nucleic acid sequence SEQ ID NO:5, SEQ ID NO:33, or SEQ ID NO:37.
 21. The hybridoma cell of claim 20, wherein said antibody-producing cell is a mammalian expression cell transfected with polynucleotides encoding immunoglobulin heavy and light chains. 